Angiostatin Overexpression in Morris Hepatoma Results in Decreased Tumor Growth but Increased Perfusion and Vascularization Kerstin Schmidt, MS1–3; Johannes Hoffend MD3; Annette Altmann, PhD2,3; Ludwig G. Strauss, MD, PhD2; Antonia Dimitrakopoulou-Strauss, MD2; Britta Engelhardt, MD1; Dirk Koczan, MD4;Jo¨rg Peter, PhD5; Thomas J. Dengler, MD6; Walter Mier, PhD2; Michael Eisenhut, PhD7; Uwe Haberkorn, MD, PhD2,3; and Ralf Kinscherf, PhD1 1Department of Anatomy and Cell Biology III, University of Heidelberg, INF 307, Heidelberg, Germany; 2Clinical Cooperation Unit Nuclear Medicine, DKFZ and University of Heidelberg, INF 280, Heidelberg, Germany; 3Department of Nuclear Medicine, University of Heidelberg, INF 400, Heidelberg, Germany; 4Department of Immunology, University of Rostock, Rostock, Germany; 5Department of Biophysics and Medical Radiation Physics, DKFZ, INF 280, Heidelberg, Germany; 6Department of Internal Medicine III, INF 410, University of Heidelberg, Germany; and 7Department of Radiopharmaceutical Chemistry, German Cancer Research Centre, Heidelberg, Germany Key Words: angiogenesis; gene therapy; PET; angiostatin; Growth of malignant tumors is dependent on sufficient blood perfusion supply. Thus, inhibition of tumor angiogenesis is emerging as a J Nucl Med 2006; 47:543–551 promising target in the treatment of malignancies. Human angiostatin (hANG) is one of the most potent inhibitors of endo- thelial cell proliferation, angiogenesis, and tumor growth in vivo. However, its mechanisms operating in vivo are not well un- derstood. Methods: To obtain more information about functional Tumor growth is angiogenesis dependent (1). Thus, changes in the angiogenic process, we established Morris hepa- inhibition of tumor angiogenesis is emerging as a promis- toma (MH3924A) cell lines expressing hANG (hANG-MH3924A). ing target in the treatment of malignancies as well as for the The effects of hANG expression on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) were mea- prevention of cancer recurrence or metastasis (2). Novel sured in coculture experiments in vitro. To evaluate changes in strategies, including endogenous antiangiogenic agents, 15 68 tumor perfusion and blood volume, H2 O and Ga-DOTA- which starve tumors by disturbing blood supply, have been albumin (DOTA is 1,4,7,10-tetraazacyclododecane-N,N,N9 $,N%- shown to be effective in preclinical models. The advantages tetraacetic acid) were used for PET studies in vivo. Additionally, of focusing on blood supply are prevention of drug resis- immunohistologic quantification of vascularization, apoptosis, tance and a highly targeted therapy of a variety of tumor and proliferation as well as gene array analyses were performed. entities (3). Human angiostatin (hANG), a cleavage product Results: Our in vitro experiments demonstrate reduced prolif- eration and increased apoptosis in HUVECs when being co- of plasminogen, is one of the most potent antiangiogenic cultured with hANG-MH3924A. In support, tumor growth of agents currently known (4). ANG exhibits profound inhi- hANG-MH3924A is diminished by 95% in vivo. However, tumor bition of endothelial cell proliferation and migration in perfusion and blood volume are increased in hANG-MH3924A vitro and angiogenesis in vivo (5). Systemic administration corresponding to an increased microvessel density. Further- of recombinant ANG inhibited angiogenesis and increased more, hANG-transfected tumors show changes in expression the apoptotic rate in several tumor models followed by of genes related to apoptosis, stress, signal transduction, and tumor regression (6). The formation of ANG implicates metabolism. Conclusion: hANG expression leads to inhibition of tumor growth, increased apoptosis, and changes in the ex- metalloproteinases or serine proteinases produced by tumor pression of multiple genes involved in stress reactions, signal cells (7) or by tumor-infiltrating macrophages (8). However, transduction, and apoptosis, which indicates a multifactorial re- the molecular mechanisms of ANG treatment in vivo remain action of tumors. An enhanced microvessel density is seen as ill defined. Therefore, monitoring of antiangiogenic ap- part of these reactions and is associated with increased perfu- proaches with functional imaging and histomorphometric sion as measured by PET. analyses are desirable to deliver information about phys- iologic effects caused by these therapeutic modalities. To Received Aug. 26, 2005; revision accepted Nov. 21, 2005. provide a noninvasive, functional relevant measure of angio- For correspondence or reprints contact: Uwe Haberkorn, MD, PhD, genesis and to obtain functional data in animals and Department of Nuclear Medicine, University of Heidelberg, INF 400, Heidelberg, 69120, Germany. humans, MRI, CT, ultrasonography, optical imaging, and 15 E-mail: [email protected] PET have been proposed (9). Especially PET using H2 O ANGIOSTATIN AND PERFUSION • Schmidt et al. 543 provides a quantitative measure of tissue perfusion—that is, in tumors with diameters between 10 and 13 mm (8–19 d after blood flow and blood volume in tumors (9). Furthermore, transplantation for WT-MH3924A and 37 and 57 d for hANG- recent data present evidence that 68Ga-DOTA-albumin MH3924A) were examined after intravenous bolus injection of 15 68 (DOTA is 1,4,7,10-tetraazacyclododecane-N,N9,N$,N%- 70–150 MBq H2 O in 0.3 mL and 5–10 MBq Ga-labeled 68 tetraacetic acid) PET can be used as a blood-pool marker in albumin [ Ga-DOTA-albumin] (same animals and session; the albumin PET scan was performed after the activity of H 15O vivo (10). The aim of this study was to evaluate the effects of 2 decayed). All PET studies were done as dynamic measurements hANG gene transfer on tumor growth and changes in perfu- 15 (20 · 3s,6· 10 s, 4 · 15 s, and 6 · 30 s for H2 O and 12 · 10 s, sion or blood volume relating in vivo measurements with PET 3 · 20 s, 4 · 30 s, and 10 · 60 s for 68Ga-DOTA-albumin) to immunohistochemistry and gene expression analysis. acquired in 2-dimensional (2D) mode using a matrix of 256 · 256 on an ECAT HR1 (Siemens/CTI) scanner (pixel size, 2.277 · MATERIALS AND METHODS 2.777 · 2.425 mm; transaxial resolution, 4.3 mm). A transmission Cell Culture and Generation of Recombinant Cells scan was done for 10 min before tracer administration with 3 rotating germanium pin sources to obtain cross sections for Cells were cultured (37°C, 95% air/5% CO2) in RPMI 1640 medium (Gibco BRL) supplemented with 292 mg/L glutamine attenuation correction. After iterative reconstruction (ordered- and 20% fetal calf serum (FCS). subsets expectation maximization; 8 subsets, 16 iterations), dy- namic PET data were evaluated following definition of volumes of hANG Gene Transfer: Northern and Western Blots interest (VOIs) as described earlier using the PMOD software MH3924A cells were transfected with hANG (pGT60hAngio- package (13). Time–activity curves were created using VOIs, statin; InvivoGen) expression vectors. The hANG protein consists which consist of several regions of interest (ROIs) over the target of 385 amino acids, including the first 4 kringle structures. Stable area. Irregular ROIs were drawn manually. Perfusion studies using 15 cell lines were generated after lipofection of MH3924A cells by H2 O were evaluated using a 1-tissue-compartment model (13). hygromycin selection (425 mg/mL) for 4 wk. RNA was isolated For the input function, we used the mean value of the VOI data using TRIZOL reagent (Roche) and messenger RNA (mRNA) obtained from the heart. ROIs were defined at 3–6 s after bolus expression was assessed by Northern blots using radioactively injection in at least 3 consecutive slices (2.4-mm thickness) labeled probes delivered by digestion from corresponding plas- surrounded by 2 slices showing the heart in each direction. This mids. Secretion of hANG protein from selected clones into cell approach was based on the findings of Ohtake et al. showing that culture media was verified by Western blots. Conditioned medium the input function can be retrieved from the image data with was produced by culture of 106 transfected Morris hepatoma cells acceptable accuracy (14). The heart weight and heart volume of a in Optimem medium (Invitrogen) for 48 h. hANG protein was 250-g rat are 1.0 g and 1.2 mL, respectively (15). This results in an isolated from medium by incubation with L-lysine-Sepharose estimated recovery error of 10%, which can be neglected for the 68 matrix followed by 3 washing steps and elution with sodium purposes of this study. The Ga-DOTA-albumin uptake was dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer. expressed as the mean standardized uptake value (SUV) between After gel electrophoresis, protein was immunoblotted on a poly- 10 and 15 min after injection when the time–activity curve reaches vinylidene fluoride membrane using polyclonal rabbit antihANG a plateau. The SUV is defined as: tissue concentration [Bq/g]/ (2 mg/mL; Oncogene) antibodies and the ECL Western Blotting (injected dose [Bq]/body weight [g]) (10). detection reagent (Amersham Biosciences). Tissue Preparation and Immunohistochemistry Measurement of Apoptotic or Proliferating Cells Rat tumors were fixed with immunohistochemistry zinc fixative Human umbilical vein endothelial cells (HUVECs) were (BD Biosciences), 4% paraformaldehyde (and embedded in par- isolated as described (11) and cultured in supplemented (4 ng/mL affin), or shock-frozen in liquid nitrogen-cooled isopentane and basic fibroblast growth factor,